WO2013188864A2 - Anticorps anti-idiotypiques anti-cd22 et leurs utilisations - Google Patents

Anticorps anti-idiotypiques anti-cd22 et leurs utilisations Download PDF

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WO2013188864A2
WO2013188864A2 PCT/US2013/046053 US2013046053W WO2013188864A2 WO 2013188864 A2 WO2013188864 A2 WO 2013188864A2 US 2013046053 W US2013046053 W US 2013046053W WO 2013188864 A2 WO2013188864 A2 WO 2013188864A2
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antibody
idiotype
antibodies
murine
binding
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WO2013188864A4 (fr
WO2013188864A3 (fr
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Shui-On Leung
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Sinomab Bioscience Limited
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Priority to US14/407,988 priority Critical patent/US9371396B2/en
Priority to EP13804606.5A priority patent/EP2861622A4/fr
Publication of WO2013188864A2 publication Critical patent/WO2013188864A2/fr
Publication of WO2013188864A3 publication Critical patent/WO2013188864A3/fr
Publication of WO2013188864A4 publication Critical patent/WO2013188864A4/fr
Priority to US15/186,999 priority patent/US10613099B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/686—Anti-idiotype
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39566—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against immunoglobulins, e.g. anti-idiotypic antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • MAb monoclonal antibody
  • Anti-idiotypic antibodies are those antibodies raised against the immunizing antibodies (Abl), and demonstrated specific binding against the idiotopes (unique antigenic determinants on the surface of the antibodies) of Abl .
  • Ab2 can be classified into three distinct groups: (1) Ab2a antibodies are those that recognize idiotopes distinct from the antigen-binding site (ABS) on primary Abl antibodies; (2) Ab2 antibodies recognize epitopes within the ABS and mimic the structure, and forming the so-called "internal image," of the nominal antigen; (3) Ab2y antibodies recognize epitopes within the ABS without the structural resemblance of the nominal antigen (Pan et al. 1995. Anti-idiotypic antibodies: biological function and structural studies. Faseb J 9:43-49).
  • ABS antigen-binding site
  • Ab2 antibodies appear to be the most interesting group of Ab2 antibodies, especially attempts to use Ab2 as surrogate antigens for the development of active vaccines against autologous and/or inert antigens such as tumor-specific or tumor associated antigens, in addition to bacterial, viral and parasitic infections (Chatterjee et al. 2001. The anti-idiotype vaccines for immunotherapy. Curr Opin Mol Ther 3(l):63-69), development of other types of Ab2 can be useful in developing assay methods that facilitate the production process and clinical evaluation of a potentially therapeutic Abl .
  • SM03 is a chimeric anti-CD22 antibody derived from the murine RFB4 antibody (Yang et al. 2006. Construction and characterization of recombinant anti-B-lymphoma chimeric antibody. Chinese J New Drugs 15(3): 186-192), and is being used in clinical trials for the treatment of non-Hodgkin's lymphoma (NHL) (Li et al. 2012. Pharmacokinetics and tolerability of human mouse chimeric anti-CD22 MAb in Chinese patients with CD22-positive non-Hodgkin's lymphoma. Austin Bioscience J 4(2):256-266).
  • NDL non-Hodgkin's lymphoma
  • SM03 targets and suppresses matured B cells
  • the antibody has expanded its indications for the treatment of other autoimmune diseases, such as, among others, Rheumatoid arthritis (RA) and Systemic Lupus Erythamatosus (SLE).
  • RA Rheumatoid arthritis
  • SLE Systemic Lupus Erythamatosus
  • SM03 was humanized using the technology of framework-patching (Liang et al. 2006. Framework-reengineering and its application in humanized antibody fSM03.
  • the framework-patched SM03 was later renamed as SM06. Both SM03 and SM06 target the same epitope of the human CD22 antigen, with comparable affinity. However, in terms of sequence and structure, the only thing SM03 and SM06 share in common is their ABSs, formed by their respective complementarity determining region (CDR) sequences.
  • CDR complementarity determining region
  • SM03 and SM06 bind to human CD22 antigen.
  • the antigen is expressed on the surface of matured B cells (Schwartz -Albiez et al. 1991.
  • CD22 antigen biosynthesis, glycosylation and surface expression of a B lymphocyte protein involved in B cell activation and adhesion.
  • the present invention is therefore directed to the use of anti-idiotypes in immunotherapy trials as diagnostic reagents for monitoring the pharmacokinetics (PK) of the administered antibody in the circulation of patients.
  • the anti-idiotype antibody can similarly be used as a positive control for HAHA, HACA or HAMA immune responses to the administered antibody. Monitoring the presence of such immune responses will influence treatment options as such immune responses may affect the clinical outcome in patients (Gruber, van Haarlem et al. 2000.
  • the human anti-mouse immunoglobulin response and the anti-idiotypic network have no influence on clinical outcome in patients with minimal residual colorectal cancer treated with monoclonal antibody C017-1A. Cancer Res. 60:1921-1926), or can be associated with undesirable hypersensitive reactions and dramatic changes in PK and biodistribution of the administered antibody.
  • Another embodiment of the present invention is to provide a general method for the evaluation of biological functions of antibodies that target internalizing antigens, including, CD22, Invariant Chain (CD74), CD33, Lewis Y antigen, etc.
  • the method includes the construction of an engineered cell line that expresses on their surface a non-internalizing fusion protein containing the anti-idiotype binding moiety.
  • the cell line that expresses surface anti-SM03 anti-idiotype antibody (or antibody fragment fusion) can be used for the evaluation of the biologic activity of SM03 and SM06 via complement-dependent cytotoxicity (CDC) or antibody directed cell cytotoxicity (ADCC) as a quality control measure.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody directed cell cytotoxicity
  • the present invention describes the generation and production of an anti-idiotype antibody that recognizes the antigen-binding site (ABS) of an anti-CD22 antibody and their derivatives (including the murine, chimeric and humanized version of the antibodies and their derivatives such as scFv, diabodies, bispecific antibodies, antibody conjugate, and antibody fusion proteins, etc.), a class collectively referred to as "the anti-CD22 antibodies".
  • the present invention further describes the use of such anti-idiotype antibodies for the development of assay methods to evaluate the identity, binding affinity, biological activities, and serum concentration of "the anti-CD22 antibodies" during clinical trials.
  • One aspect of the invention is to provide anti-idiotype antibodies specific for "the anti-CD22 antibodies”. Another aspect of the invention is to provide anti-idiotype antibodies which bind to the variable region of "the anti-CD22 antibodies.” Yet another aspect of the invention is to provide anti-idiotype antibodies which bind to the ABS of "the anti-CD22 antibodies.” Similarly, another aspect of the invention is an anti-idiotype antibody which blocks the binding of an anti-CD22 MAb to its nominal (CD22) antigen. Another aspect of the invention is to provide an anti-idiotype antibody which specifically binds RFB4, a murine antibody. Similarly, a further aspect of the invention is to provide an anti-idiotype antibody which specifically binds SM03, a chimeric antibody.
  • Yet a further aspect of the invention is to provide an anti-idiotype antibody which specifically binds SM06, a humanized antibody using the framework-patching technology.
  • the anti-idiotype antibodies provided for in this invention can be selected from the group consisting of a murine MAb, a chimeric antibody, a human antibody, a humanized antibody, a single chain antibody, or a disbud, and other forms of fusion proteins.
  • Another aspect of the invention is to provide a transfected cell line capable of producing an ant-idiotype antibody specific for "the anti-CD22 antibodies.”
  • a further aspect of the invention is to provide a cell line producing an anti-idiotype antibody which is specific for "the anti-CD22 antibodies” selected from the group consisting of RFB4 (murine antibody), SM03 (chimeric antibody) and SM06 (framework-patched antibody).
  • Another embodiment of the invention is to provide methods for detecting the ability of an anti-idiotype antibody to inhibit the binding of antibody to antigen.
  • a further aspect of the invention is to provide methods for detecting the ability of anti-idiotype antibodies to capture and detect bound idiotype antibody
  • Another aspect of the invention is to provide methods for detecting the ability of anti-idiotype antibody to bind to "the anti-CD22 antibodies.”
  • Another aspect of the invention is to provide methods of detecting the amount of "the anti-CD22 antibodies" in sample serum.
  • the present invention is also directed against a method to detect HAMA, HACA and HAHA responses using the antibody of the invention.
  • Another aspect of the present invention is to provide an engineered cell line with surface expression of the binding moiety of the anti-idiotype antibody.
  • a further aspect of the invention is directed against a method to assess the biological activities of "the anti-CD22 antibodies" using the engineered cell lines as target cells for CDC and/or ADCC assays.
  • Figure 1 depicts the specific binding of different scFv Phages (Phages #1-3) against murine (RFB4)(p ), chimeric (SM03)( ⁇ ) and framework-patched (SM06)( ⁇ ) anti-CD22 antibodies.
  • the phages do not exhibit significant binding against a control anti-TNF antibody (chimeric) ⁇ ) and BSA( gg).
  • FIG. 2 depicts the complementarity determining region (CDR) sequences of three selected scFv phages (Phage #1-3) interacted with RFB4, SM03 and SM06 (murine, chimeric and humanized versions) but not with other antibody (anti-TNF) and control protein (BSA). Since the only sequence in common between murine RFB4, SM03, and SM06 would be in the CDR sequences, or the antigen binding region, of the target antibodies, these results suggested that all three selected scFv phages were specific for the idiotype of SM03.
  • CDR complementarity determining region
  • Figure 2A depicts the heavy chain CDR sequences, SEQ ID NOs: 1-3
  • Figure 2B depicts the light chain CDR sequences, SEQ ID NOs: 4-6, for the scFv phages #1-3, referred to as CDR1, CDR2, and CDR3, respectively.
  • Antibodies are identical, except for a single amino acid sequence difference (underlined) in the CDR3 region of the light chain in phage #2 (CDR3-2).
  • Figure 3 depicts the amino acid sequence (single letter code, SEQ ID NO: 7) of scFv isolated from phage #3 which showed specific binding to RFB4, SM03 and SM06. CDR sequences are boxed.
  • the configuration of the scFv is VH-linker-VL.
  • the linker sequence (shown in italics) used is G 4 -S-G-S-G-S-S-G 4 (SEQ ID NO: 16).
  • FIG. 4 graphically demonstrates how single-chain antibody (scFv) derived from phage #3 can effectively inhibit the binding of SM03 to Raji cells in a flow-cytometry assay.
  • scFv single-chain antibody
  • Figure 5 depicts the pharmacokinetic profile of a lymphoma patient treated with SM03 (360 mg/m 2 , i.v., once a week, four weeks).
  • the serum concentration of SM03 was determined by ELISA with immobilized scFv from phage #3 as the capture antibody.
  • Figure 6 depicts the amplifiable DNA vector for the expression of anti-idiotype antibody as murine IgG2a/kappa immunoglobulins.
  • Figure 7 depicts cDNA sequences for the heavy (Fig. 7A) and light (Fig. 7C) immunoglobulin chains of the "anti-idiotype mlgG" expressed in clone AE6 using standard RT-PCR and Sanger's dideoxynucleotide sequencing procedures.
  • the deduced amino acid sequences in single-letter code of the respective chains are also shown, in Fig. 7B and 7D respectively.
  • Figure 8 depicts the results of SDS-PAGE electrophoresis showing the immunoglobulin chains of the "anti-idiotype mlgG" expressed in clone AE6 under reducing and non -reducing conditions.
  • Figure 9 demonstrates how SM03, but not other antibodies (anti-CD20, anti-CD 147 and anti-TNF antibodies), specifically binds to the "anti-idiotype mlgG" purified from clone AE6.
  • Figure 10 depicts the results of a flow cytometry study showing that the "anti-idiotype mlgG" can effectively block the binding of SM03 on CD22 expressed on the surface of Raji cells.
  • Figure 11 demonstrates how murine RFB4, chimeric SM03 and framework-patched SM06 can effectively compete with SM03-HRP conjugates for the binding to the "anti-idiotype mlgG.”
  • Figure 12 depicts the pharmacokinetic profile of Lupus patients treated with SM03.
  • the serum concentration of SM03 was determined by ELISA with immobilized "anti-idiotype mlgG" purified from clone AE6 as the capture antibody.
  • Figure 13 depicts the structure and amino acid sequence (SEQ ID NO: 12) of the heavy chain of the "anti-idiotype mlgG" fused to the transmembrane (TM) sequence of murine IgD.
  • Figure 14 depicts the structure and amino acid sequence (SEQ ID NO: 13) of the "anti-idiotype mlgG" carrying murine IgD heavy chain (TM sequence underlined).
  • Figure 15 depicts the structure and amino acid sequence (SEQ ID NO: 14) of the "anti-idiotype mlgG” Fd-glycophorin A fusion protein.
  • Figure 16 depicts the structure and amino acid sequence (SEQ ID NO: 15) of the "anti-idiotype mlgG” Fd-GPI fusion.
  • Figure 17 presents a comparison of surface expression of "anti-idiotype mlgG" in the form of murine IgD, Fab-glycophorin A fusion, and Fab-GPI-fusion proteins on transfectoma cell lines.
  • Figure 18 demonstrates how complement-dependent cytotoxicity (CDC) is induced by SM03 against transfectoma cell lines with surface expression of "anti-idiotype mlgG" in the form of murine IgD, Fab-glycophorin A fusion, and Fab-GPI-fusion proteins.
  • CDC complement-dependent cytotoxicity
  • the anti-CD22 MAbs described herein refer to murine, chimeric, humanized (framework-patched) antibodies and their derivatives, including but not limited to, antibody fragments (Fab, Fab', F(ab') 2 ), scFv, diabodies, bispecific antibodies, and antibody fusion proteins, etc., and are collectively referred to hereinafter as "the anti-CD22 antibodies.”
  • the present invention provides an anti-idiotype antibody whose binding moiety interacts specifically to the variable regions of the "anti-CD22 antibodies”.
  • an anti-idiotype antibody whose binding moiety interacts specifically to the ABS of "the anti-CD22 antibodies.”
  • One embodiment of the invention is an anti-idiotype antibody, which effectively inhibits the binding of "the anti-CD22 antibodies” to its natural ligand (human CD22).
  • another aspect of the invention is to provide an anti-idiotype antibody, which binds the anti-CD22 MAb, RFB4.
  • RFB4 the anti-idiotype antibody, which binds the anti-CD22 chimeric antibody, SM03 (Yang et al. 2006. Construction and characterization of recombinant anti-B-lymphoma chimeric antibody. Chinese J New Drugs 15(3): 186-192).
  • an anti-idiotype antibody which binds the anti-CD22 antibody humanized by framework-patching, SM06 (also named as fSM03) (Liang et al. 2006. Framework-reengineering and its application in humanized antibody fSM03. Chinese J New Drugs 15(21): 1832-1836).
  • the anti-idiotype antibodies provided for in this invention can be selected from the group consisting of a MAb of murine isotypes, a chimeric antibody, a human antibody, a humanized or framework-patched antibody, a single chain antibody, diabody, bispecific antibody and other antibody fusion proteins.
  • Another embodiment of the present invention is to provide a transfectoma capable of producing an anti-idiotype antibody specific for "the anti-CD22 antibodies”.
  • immunoglobulin refers to a protein molecule consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Full length immunoglobulin "light chains” (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH 2 -terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus.
  • variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).
  • antibody is used broadly to refer to both antibody molecules and its derivatives. Such derivatives contain at least one variable region from either a heavy or light immunoglobulin chain, and should encompass molecules such as antibody fragments in the form of F(ab')2, Fab, Fab', Fd, Fabc, scFv, diabodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains, bispecific antibodies and other molecules, and the like.
  • variable region as used herein in reference to immunoglobulin molecules has the ordinary meaning given to the term by the person skilled in the art of immunology. Both antibody heavy and light chains may be divided into a “variable region” and a "constant region". The person skill in the art may readily distinguish a variable region from a constant region by reference to standard tests describing antibody structure, e.g., Kabat et al. 1991. "Sequences of Proteins of Immunological Interest: 5 th Edition" US Department of Health and Human Services, US Government Printing Office.
  • chimeric antibody refers to a reengineered protein molecule with the heavy and light chain variable region sequences derived from non-human species, while the constant region sequences are derived from human immunoglobulin.
  • humanized antibody refers to a reengineered protein molecule with all its CDRs (complementarity determining regions) derived from the variable region sequences of immunoglobulin that is of non-human species origins, while the majority of the remainder of the sequences are derived from a human immunoglobulin.
  • idiotype refers to the segment of an antibody molecule that determines its specificity for antigen.
  • the idiotype is located in the Fab region, and its expression usually requires participation of the variable regions of both heavy and light chains that form the antigen-combining site.
  • antigen-binding site refers to the region(s) of an antibody molecule to which a ligand actually binds, and is derived from an antibody; the term “antigen-binding site” include antibody heavy chain variable domains (VH) and/or an antibody light chain variable domains (VL), or pairs of VH/VL, and can be derived from whole antibodies or antibody fragments such as single chain Fv, a VH domain and/or a VL domain, Fab, or (Fab)2.
  • VH antibody heavy chain variable domains
  • VL antibody light chain variable domains
  • each of the antigen-binding sites comprises an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL), and preferably is formed by a pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
  • isotype refers to antigens that determine the class or subclass of heavy chains or the type and subtype of light chains of immunoglobulin molecules.
  • isotypes of IgG are designated IgGl, IgG2, IgG3 and IgG4.
  • CD22 antigens are expressed on the surface of mature B cells and malignant B cells (Droken et al. 1989. B-cell antigens: CD22. In Knapp et al.,eds. Leucocyte Typing IV: White cell differentiation antigens. New York, Oxford University Press. P.63-64). CD22 is a regulatory molecule that prevents the over activation of the immune system and the development of autoimmune diseases (Hatta et al. 1999. Identification of the gene variations in human CD22. Immunogenetics 49(4):280-286).
  • One of "the anti-CD22 antibodies” described herein is derived from the murine antibody RFB4.
  • SM03 and SM06 versions like their murine counterpart, specifically target the B epitope of the human CD22 antigen.
  • Clinical trials for the treatment of B-cell lymphoma and other autoimmune diseases with SM03 are underway.
  • anti-idiotype antibodies were generated and characterized for suitability as ELISA reagents for measuring "the anti-CD22 antibodies” in patient sera samples.
  • the anti-idiotype antibody thus generated can be used for the development of ELISA reagents for measuring HACA or HAHA responses, either as control antibodies or as diagnostic agent to evaluate the presence of competing antibodies in patient serum.
  • the anti-idiotype antibody was generated from mice immunized with SM03 using phage display technologies. Specifically, messenger RNA were isolated from splenocytes of mice immunized with the anti-CD22 chimeric antibody, SM03, and degenerate variable region flanking primers used to amplify heavy and light chain variable region sequences which were subsequently incorporated into a scFv phage display libraries following standard procedure. Upon several rounds of panning with SM03 and RFB4, antibody phages that showed binding against SM03, RFB4 and SM06 were identified, and the variable region sequences of the respective phages elucidated. The variable region sequences of the antibody phage that demonstrated the highest affinity against murine, chimeric and humanized forms of "the anti-CD22 antibodies" were selected.
  • the anti-idiotype antibody sequence was over-expressed in E. coli as scFv inclusion bodies, which were subsequently denaturated and refolded; the active scFv was used to develop ELISA reagents for the detection of serum levels of SM03 in clinical trial.
  • refolded scFv anti-idiotype antibody was used to coat ELISA plate, and serum from patients treated with SM03 was added, incubated, and the wells washed.
  • SM03 present in the serum would bind to the coated anti-idiotype scFv, and could be revealed by the addition of HRP-conjugated goat anti-human Fc-specific antibody (Jackson ImmunoResearch, West Grove, PA).
  • the anti-idiotype scFv tends to be unstable, and production from bacterial inclusion bodies could result in variations in the quality of the denatured and refolded proteins, making the results inconsistent.
  • the unstable nature of the anti-idiotype scFv it would make storage, and therefore preparation of validated batches for the anti-idiotype scFv difficult.
  • Full immunoglobulin molecules are known to be stable, when stored under appropriate conditions, for months, if not years, without significant changes in the quality of the antibody.
  • the variable region sequences of the anti-idiotype scFv were used to construct a full length immunoglobulin molecule.
  • the chimeric or humanized versions of "the anti-CD22 antibodies” both carry human IgGl and kappa constant region sequences, they can be detected by standard HRP-conjugated anti -human Fc antibodies (or similar conjugates).
  • anti-idiotype immunoglobulin should not carry constant region sequences that might cross-react with the detecting conjugates.
  • Murine IgG2a/kappa constant regions do not cross react with anti-human Fc antibodies, and are therefore chosen for constructing full immunoglobulin for the anti-idiotype antibody. It should be noted, however, constant region sequences of different isotypes and from different species can also be used. Cell lines that produced over 3C ⁇ g/ml of the anti-idiotype antibody with the murine IgG2a/kappa ("anti-idiotype mlgG") were generated.
  • DHFR dihydrofolate reductase
  • Serum anti-CD22 antibodies that interacted with the coated "anti-idiotype mlgG” was revealed by the addition of HRP-conjugated goat anti-human-Fc-specific antibody (Jackson ImmunoResearch). Since murine constant region of the "anti-idiotype mlgG" do not cross react with the detecting HRP conjugate, the assay does not result in problems with background signals. The assay method was proven to be highly reproducible, sensitive, and specific.
  • One other aspect of the current invention is to make use of the anti-idiotype antibody to construct cell lines that express the SM03-specific binding moieties of the anti-idiotype antibody in different forms or as fusion proteins on the cell surface in a non-internalizing mode.
  • the cell lines developed can be used for the establishment of biological assays to evaluate the bioactivities of SM03 and/or its derivatives such as RFB4 and SM06, etc.
  • the method is meant to confer a general applicability for the evaluation of biological activities of antibodies that bind to internalizing surface antigens.
  • mice of ⁇ 6 weeks old were immunized intra-peritoneally with 100 g of SM03, which was emulsified in 200 ⁇ of complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO) following a standard immunization protocol (Harlow and Lane, 1988. In Antibodies: A Laboratory Manual. New York, Cold Spring Harbor Laboratory). Secondary and tertiary immunizations were carried out at intervals of 14, and 35 days by intra-peritoneal injection of 100 g of SM03 emulsified in 200 ⁇ of incomplete Freund's adjuvant (Sigma-Aldrich).
  • RNA from the spleen of immunized mice was extracted for cDNA preparation (Superscript II, Invitrogen, Grand Island, NY).
  • PCR amplification of immunoglobulin variable regions was performed using degenerate primers as described in Cheng et al. (Cheng et al. 2005. Cross-reactivity of antibody against SARS-coronavirus nucleocapsid protein with IL-11. Biochem Biophys Res Commun 338(3): 1654-60).
  • Phage library displaying scFv was constructed using Amersham's recombinant phage antibody system (Amersham, Piscataway, NJ) according to manufacturer's specifications.
  • phages at a concentration of 10 12 were biopanned against equal amounts (100 ⁇ g/ml) of SM03 or murine RFB4 (Ancell) in carbonate coating buffer (15 mM Na 2 C0 3 , 35 mM NaHCC>3, pH 9.6). After incubation at room temperature for 2 h with gentle shaking, bound scFv-phages were eluted by incubation at room temperature for 10 min with 100 ⁇ of 0.1 M glycine-HCl, pH 2.2, followed by neutralization with 10 ⁇ of 1 M Tris-HCl, pH 8.0. The selection process was repeated four times with the input and output phage titers at each round recorded.
  • Phages that survived panning were rescued.
  • the binding specificities of selected phages were evaluated by Phage-ELISA using the target anti-CD22 antibodies RFB4, SM03, and framework-patched (humanized) SM06 and other control antibodies as antigens.
  • a 96-well ELISA plate (NUNC, Roskilde, Denmark) that were coated with RFB4, SM03, and SM06, chimeric anti-TNF antibody, and BSA (coated with 50 ⁇ of carbonate coating buffer, pH 9.6, containing 1 ⁇ g of antibody/BSA; incubated at 4°C overnight; washed three times with 200 ⁇ of borate washing buffer, pH8.0; and blocked with borate washing buffer, pH8.0 at 37°C for 1 h), three clones of phages that showed the highest degree of binding to SM03 (100 ⁇ of culture supernatant) were added for incubation at 37°C for 1 h.
  • HRP horse radish peroxidase
  • Inclusion body containing scFv was collected after IPTG induction, and was denatured (6 M guanidine HC1 in 20 mM sodium phosphate and 0.5 M NaCl, pH 7.4), refolded, and purified using HiTrap Chelating HP column according to the manufacturer's specifications (Amersham). Briefly, denatured scFv containing a His tag were bound to HiPtrap Chelating HP column with Ni 2+ added. Decreasing concentration (in gradient) of guanidine HC1 in 20 mM sodium phosphate and 0.5 M NaCl, pH 7.4, was applied until all the guanidine HC1 in the column was cleared.
  • the column was washed with several bed volumes of 5 - 40 mM imidazole (Sigma) in 20 mM sodium phosphate and 0.5 M NaCl, pH 7.4. The eluted samples were pooled and the protein examined by SDS-PAGE electrophoresis (not shown).
  • soluble scFv was used to compete with cell surface CD22 (Raji cells) for binding to SM03.
  • soluble scFv derived from Phage #3 effectively inhibited the binding of SM03 onto CD22 antigen on the surface of Raji cells, as revealed by a significant reduction in fluorescence when evaluated by flow cytometry ( Figure 4).
  • the single-chain scFv from Phage #3 had demonstrated specificity against SM03, and can therefore be used for the development of assay method for the evaluation of blood levels of SM03 in patients treated with the anti-CD22 antibody, especially in clinical trials where pharmacokinetic (PK) studies were required.
  • the scFv from Phage #3 was prepared as described above, and used to coat 96-well ELISA plates (NUNC). The plate was then blocked with BSA, washed, and blood samples collected at different time points from patients treated with SM03 were added.
  • TMB Tetramethyl Benzidine
  • Figure 5 showed a typical PK profile of SM03 evaluated using the aforementioned assay method in a lymphoma patient treated with the anti-CD22 antibody.
  • SM03 was administered at 380 mg/m 2 , once a week for four weeks. Blood samples were collected at different time points before and after SM03 administration.
  • the expression vector could be used to transfect a variety of mammalian host cell lines, including, without limitation, Chinese hamster ovary (CHO) cells, murine myeloma SP2/0 or NSO, baby hamster kidney (BHK) cells, human embryonic kidney 293 (HEK 293) cells, African green monkey kidney COS cell line, etc.
  • SP2/0 cells were transfected with the expression vector by electroporation following standard procedure. Clones surviving methotrexate (MTX) selection were tested for antibody expression. Clone T081210AE6 (AE6) was tested positive, and showed the highest level of expression for murine IgG.
  • Anti-idiotype mlgG purified from clone AE6 demonstrated direct binding to SM03
  • Clone AE6 was expanded and antibody was purified from the culture supernatant by Protein A chromatography following standard procedure.
  • the purified antibody was stored in PBS at 4°C.
  • Figure 8 showed the SDS-PAGE profile of the purified antibody under reducing and non-reducing conditions.
  • the purified "anti-idiotype mlgG" derived from clone AE6 was used to coat ELISA plate following standard procedures. To the wells of the coated plate, 60 ⁇ of SM03, and other irrelevant control antibodies such as anti-CD20 (humanized), anti-CD 147 (chimeric), and anti-TNF (infliximab) antibodies at various concentrations were added. After an incubation period of 1.5 h at room temperature, the plate was washed three times with PBS, and HRP-conjugated goat-anti-mouse Fc-specific antibody (l :5000)(Jackson ImmunoResearch) were added.
  • HRP-conjugated goat-anti-mouse Fc-specific antibody l 5000
  • Anti-idiotype mlgG from clone AE6 binds specifically to the antigen-binding site (ABS) sequences shared by "the ant-CD22 antibodies "
  • the "anti-idiotype mlgG" from clone AE6 binds specifically against the murine, chimeric and humanized form of the anti-CD22 antibody.
  • ABS antigen-binding site
  • SM03 antibodies were conjugated with HRP (SM03-HRP) by TJ Biotechnologies Limited (Tianjin, China).
  • HRP SM03-HRP
  • TJ Biotechnologies Limited TJ Biotechnologies Limited (Tianjin, China).
  • SM03-HRP at 1 :4000 dilution was mixed with varying concentrations of competing antibodies, including RFB4, SM03 and SM06, and other irrelevant control antibodies.
  • the mixtures were added into the wells of ELISA plate coated with the "anti-idiotype mlgG".
  • the level of binding of SM03-HRP to the "anti-idiotype mlgG" coated on the ELISA plate after competition was revealed by TMB assays following the manufacturer's specifications (Invitrogen).
  • an ELISA assay can be developed to evaluate the concentration of R.FB4, SM03 and SM06 in patient sera during clinical trials. While the example below described the development of an assay to evaluate serum levels of SM03, the method is generally applicable to other members of "anti-CD22 antibodies.” Briefly, each well of Microliter 96-well plates was coated with 0.4 ⁇ / ⁇ of the "anti-idiotype mlgG” in 50 ⁇ , PBS, pH 7.4 (4 " C , overnight) and was then blocked with 1% BSA for 2 h at room temperature. Serum samples from patients treated with SM03 were diluted with PBS at different concentrations.
  • exogenous SM03 at various known concentrations were diluted with PBS in the presence of normal human serum at a final concentration of 0.1%.
  • 50 ⁇ of serum samples at duplicates were added into the wells of the coated plates, which were subsequently incubated for 2h at room temperature.
  • HRP-conjugated goat anti-human IgG Fc antibody (1 :4000) Jackson ImmunoResearch was loaded and incubated for I h at room temperature. The reaction was visualized at ⁇ 45 ⁇ , after the addition of 50 ⁇ !
  • TMB chromogenic substrate 3,3',5,5 ' -tetramethylbenzidine
  • Figure (12) shows a typical PK profile of a patient treated with SM03, established using the ELISA assay method developed as described above.
  • binding properties manifested as affinity and specificity
  • ADCC biological response
  • bioassays Since "the anti-CD22 antibodies” bind to human CD22 antigen, which is known to be internalized at a rapid rate (Leung et al. 1995. Construction and characterization of a humanized, internalizing B (CD22)-specific, leukemia/lymphoma antibody, LL2. Mol. Immunol.
  • SM03 as well as other anti-CD22 antibodies, fails to demonstrate the ability to induce CDC reactions in vitro (Liang et al. 2006; Carnahan et al. 2007.
  • Epratuzumab a CD22-targeting recombinant humanized antibody with a different mode of action from Rituximab. Mol. Immunol. 44:1331-1341).
  • the anti-CD22 antibodies bound on the surface of CD22 + cells would not stay on the cell surface long enough to allow the Fc portion of the antigen-bound antibodies to interact with the complement, presumably Clq, for the initiation of a cascade of events leading to cell lyses.
  • the binding moiety of the anti-idiotype antibody in the present invention is therefore reengineered to allow for expression on the cell surface as a non-internalizing membrane-bound protein. It is done by one of the following ways:
  • a synthetic DNA sequence encoding a portion of the C-terminal end for the "anti-idiotype mlgG" CH3 domain fused to the TM domain of murine IgD was generated.
  • the DNA sequence was flanked by BsrGl and Eagl site to facilitate cloning into the corresponding cloning sites found on the original murine IgG2a constant region.
  • the sequence of the synthetic DNA is shown below (cloning restriction sites underlined).
  • the sequence encodes the last 41 amino acids of the original murine IgG2a fused in frame to the TM(IgD) sequence and is shown below : rGrACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCA
  • the above sequence was cloned into the corresponding cloning sites in the original IgG2a sequence, replacing the C-terminal portion of the IgG2a CH3 domain sequence with a fusion gene containing the same portion of the replaced IgG2a CH3 sequence fused in-frame to the TM sequence of murine IgD.
  • the expressed protein would contain the heavy chain IgG2a immunoglobulin fused to the TM sequence derived from murine IgD immunoglobulin (VH-CHl-hinge-CH2-CH3-TM) (See Figure 13 and SEQ ID NO: 12).
  • the original IgG2a constant region sequence of the "anti-idiotype mlgG” was replaced with that of membrane bound murine IgD.
  • the amino acid sequence of murine IgD with the TM sequence (underlined) is shown below:
  • the DNA sequence encoding the cDNA of the membrane bound form of murine IgD was chemically assembled by standard oligonucleotide synthesis. Cloning restriction sites (Xhol and Eagl) were introduced to facilitate cloning into the corresponding sites of the original murine IgG2a expression vector. The sequence of the synthetic DNA is shown (cloning restriction sites underlined). Coding sequence is shown in uppercase, and non-translated sequence in lowercase.
  • Iga expression vector (not shown) was co-transfected with the IgD-TM expression vectors into SP2/0 cell using standard electroporation techniques. Cells transfected with the plasmid were selected in the presence of methotrexate conferred by the dihydrofolate reductase (DHFR) gene on the plasmids by standard methods.
  • DHFR dihydrofolate reductase
  • Cells surviving selection were tested for surface expression of the anti-idiotype specificity by cell-based ELISA assay. Briefly, 50 of SM03 at 10 g/ml were added to lxl O 6 of transfected cells (washed 3X with PBS). The mixture was incubated for 1 h at 4°C, and washed 3X with PBS. 50 of HRP-conjugated goat anti-human IgG Fc-specific antibody (Jackson ImmunoResearch) at a dilution of 1 : 1000 were added into the cells, and were incubated for 1 h at 4°C.
  • HRP-conjugated goat anti-human IgG Fc-specific antibody Jackson ImmunoResearch
  • the transmembrane region of Glycophorin A from the red-cell membrane was fused to the C-terminal end of the murine IgG2a hinge region of the "anti-idiotype mlgG.”
  • the antibody fragment fusion was expected to be expressed on the surface of transfected cells in the form of Fab anchored on the cell surface via the transmembrane region of the Glycophorin A.
  • the amino acid sequence of the portion of Glycophorin A sequence (including the transmembrane region: boxed) used for fusion to the anti-SM03 antibody fragment is shown below:
  • the expressed protein would contain the heavy chain Fd region of the "anti-idiotype mlgG" fused to the transmembrane and cytoplasmic regions of Glycophorin A protein (VH-CHl -hinge-Glycophorin A transmembrane + cytoplasmic region) ( Figure 15 and SEQ ID NO: 14).
  • An expression vector for the surface expression of the "anti-idiotype mlgG" Fd fragment - Glycophorin A fusion protein was constructed. Briefly, a DNA sequence encoding portions of the murine IgG2a CHI -hinge region fused to the C-terminal portions (including the transmembrane region sequence) of the Glycophorin A sequence was chemically synthesized by standard oligonucleotide synthesis. The sequence contains in-frame cloning sites Xhol and Eagl (underlined).
  • the synthetic sequence was cloned into the corresponding cloning sites of the anti-SM03 IgG2a expression vector ( Figure 6), replacing the CH2-CH3 domain coding sequences of the original IgG2a sequences, using standard techniques in molecular biology.
  • the synthetic sequence is shown below.
  • GGAGATCGAGAACCCCGAGACCAGCGACCAGTAAgtgcgacggccggc SEQ ID NO: 23.
  • Plasmid DNA for the expression vector for the "anti-idiotype mlgG" Fab-glycophorin A fusion protein was linearized and transfected into mouse SP2/0 cells. Cells transfected with the plasmid were selected in the presence of methotrexate conferred by the dihydrofolate reductase (DHFR) gene on the plasmids by standard methods. Cells surviving selection were tested for surface expression of the anti-idiotype Fab-glycophorin A fusion first by Cell-based ELISA as described above (see Table 2 hereinbelow).
  • DHFR dihydrofolate reductase
  • SM03 was used as the primary antibody, and FITC conjugated goat-anti-human Fc-specific antibody as the detecting (secondary) antibody. Briefly, 5X10 5 of the transfected cells were incubated with 1 g of SM03 in a final volume of 100 ⁇ of PBS supplemented with 1% FCS and 0.01% (w/v) sodium azide (PBS-FA). The mixtures were incubated for 30 minutes at 4°C and washed three times with PBS to remove unbound antibodies.
  • PBS-FA sodium azide
  • the binding levels of SM03 to the transfected cells were assessed by the addition of a 20x diluted FITC-labeled, goat anti-human IgGl , Fc fragment-specific antibodies (Jackson ImmunoResearch) in a final volume of 100 ⁇ in PBS-FA, and incubating for 30 minutes at 4°C. The mixture was washed three times with PBS and fluorescence intensities were measured by FACSCAN analysis (Becton Dickinson). Results indicated that anti-idiotype Fab-glycophorin A fusion proteins were effectively expressed on the cell surface of the transfected myeloma cells ( Figure 17).
  • GPI-signal derived from LDL receptor attached to a DAF hydrophobic domain was fused downstream of the IgG2a hinge region of the "anti-idiotype mlgG.”
  • the fusion protein is expected to be expressed on the surface of transfected cells in the form of Fab attached on the cell surface via the GPI anchor.
  • the amino acid sequence of the portion of the GPI-signal (boxed) derived from LDL receptor attached to a DAF hydrophobic domain is shown below.
  • GPI sequence LTTSGIVTMSHQALG - FTLTGLLGTLVTMGLLT (SEQ ID NO: 24).
  • An expression vector for the surface expression of the "anti-idiotype mlgG" Fab-GPI-DAF fusion protein was constructed. Briefly, a DNA sequence encoding portions of the murine IgG2a CHI -hinge region fused to the GPI signal-DAF sequence was chemically assembled by standard oligonucleotide synthesis. The sequence contains in-frame cloning sites Xhol and Eagl (underlined).
  • Plasmid DNA for the expression vector for the "anti-idiotype mlgG” Fab-GPI-DAF fusion protein was linearized and transfected into mouse SP2/0 cells. Cells transfected with the plasmid are selected in the presence of methotrexate conferred by the dihydrofolate reductase (DHFR) gene on the plasmids by standard methods. Cells surviving selection were tested for surface expression of the "anti-idiotype mlgG" Fab-GPI-DAF fusion first by Cell-based ELISA as described above (see Table 3 hereinbelow).
  • DHFR dihydrofolate reductase
  • SM03 was used as the primary antibody, and FITC conjugated goat-anti-human Fc-specific antibody as the detecting (secondary) antibody. Briefly, 5X10 5 of the transfected cells were incubated with 1 g of SM03 in a final volume of 100 ⁇ of PBS supplemented with 1% FCS and 0.01% (w/v) sodium azide (PBS-FA). The mixtures were incubated for 30 minutes at 4°C and washed three times with PBS to remove unbound antibodies.
  • PBS-FA sodium azide
  • the binding levels of SM03 to the transfected cells were assessed by the addition of a 20x diluted FITC-labeled, goat anti-human IgGl, Fc fragment-specific antibodies (Jackson ImmunoResearch) in a final volume of 100 ⁇ in PBS-FA, and incubating for 30 minutes at 4°C. The mixture was washed three times with PBS and fluorescence intensities were measured by FACSCAN analysis (Becton Dickinson). Cell clones that demonstrated the highest level of fluorescent intensity were expanded for further tests.
  • Transfected cells demonstrated to have surface expression of the anti-idiotype IgD-TM, the "anti-idiotype mlgG” Fab-glycophorin A and Fab-GPI-DAF fusion proteins were adjusted to a density of 2xl0 6 /ml.
  • 50 ⁇ of the cells were added into each well of a 96-well microtiter-plate.
  • Guinea pig serum (GPS) obtained from Cedarlane (Burlington, Ontario, Canada) in lyophilized form was reconstituted with 1 ml of culture media (100% GPS).
  • 50 ⁇ of SM03 at various concentrations containing 10% GPS were added into wells containing the transfected cells.

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Abstract

La présente invention concerne la génération d'un anticorps anti-idiotypique monocaténaire Fv (scFv) spécifique des versions murine (RFB4), chimère (SM03) et humanisée (SM06) d'un anticorps anti-CD22 (les anticorps anti-CD22). La présente invention, concerne, en outre, la construction d'une immunoglobuline murine IgG2a/kappa porteuse des séquences de la région variable desdites séquences scFv anti-idiotypiques. La présente invention concerne, par ailleurs, une lignée cellulaire capable de produire un anticorps murin anti-idiotypique spécifique des anticorps anti-CD22. La présente invention concerne aussi un procédé d'identification et d'évaluation de l'activité et de la concentration des anticorps anti-CD22. La présente invention concerne, de plus, un procédé d'évaluation de la concentration sérique en anticorps anti-CD22 utilisés en clinique. La présente invention concerne un procédé de détection des réponses HAMA, HACA et HAHA chez des patients traités par les anticorps anti-CD22. La présente invention concerne, plus précisément, l'établissement d'une lignée cellulaire exprimant la concentration en anticorps de l'invention à sa surface; ladite lignée cellulaire exprimant à sa surface des anticorps anti-idiotypiques ou des fragments d'anticorps sera utilisée en tant que lignée cellulaire cible en vue de l'évaluation de l'activité fonctionnelle des anticorps anti-CD22 via la cytotoxicité dépendant du complément (CDC) et/ou la cytotoxicité cellulaire dépendant des anticorps (ADCC).
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WO2022204070A1 (fr) 2021-03-22 2022-09-29 Juno Therapeutics, Inc. Procédés de détermination de la puissance d'une composition de cellules thérapeutiques
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WO2023168305A1 (fr) 2022-03-01 2023-09-07 Exuma Biotech Corp. Particules virales comprenant de l'hyaluronidase liée à une membrane

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